Abstract Heart failure afflicts millions of Americans, and its incidence is only increasing. Unfortunately, heart failure is extremely debilitating, and without intervention, patients will eventually die. To date, the only cure for heart failure is transplant. In the majority of cases, heart failure is caused by an ischemic injury which leads to cardiomyocyte dysfunction and death. This results in reduced myocardial contractility and prevents adequate tissue perfusion. Metabolic dysfunction and cardiomyocyte death are major contributing factors in the initiation and progression of heart failure. Nicotinamide adenine dinucleotide (NAD) is a coenzyme that is known to contribute to both energy generation and pro-survival signaling. NAD is decreased in both pressure-overload induced and dilated cardiomyopathy induced heart failure. However, it is still not defined whether this occurs in the more common ischemic HF, or if the NAD reduction is causal or merely correlative. We hypothesize that mitochondrial NAD depletion is a major initiator of metabolic derangements and cardiomyocyte death that lead to the progression of ischemic heart failure. Our goal in this proposal is first to determine how the nuclear/cytosolic and mitochondrial NAD pools are altered during ischemic heart failure. We will also determine how NAD depletion contributes to the metabolic shift in substrate utilization observed by the injured heart. Additionally, we will evaluate if the depletion of NAD is the principal mechanism for the initiation and progression of heart failure. We will utilize a genetic mouse model that specifically depletes NAD in the heart to determine if decreasing NAD alone without any other stress can result in cardiac dysfunction. In addition to depleted NAD, HF results in a dramatic increase in nicotinamide kinase 2 (NRK2) expression. NRK2 is a kinase that can generate NAD from nicotinamide riboside (NR) independent of nicotinamide phosphoribosyltransferase (Nampt), the enzyme that catalyzes the rate- limiting step in the salvage pathway for NAD synthesis, which is thought to be the primary mechanism for NAD generation in cardiomyocytes. However, it is not well established if and how NR can prevent the progression of ischemia-induced heart failure. We speculate that the increase in NRK2 expression helps to synthesize additional NAD to compensate for the observed depletion. However, NR is not thought to be a highly abundant metabolite. For this proposal, we plan to examine if NR supplementation can improve the outcome in ischemic heart failure by improving metabolic performance and increasing myocardial regeneration. Also, to understand why NRK2 expression is increased during heart failure, we plan to subject NRK2 knockout mice to ischemia- induced heart failure and determine how loss-of NRK2 contributes to the progression of HF. This will allow us to conclude the pathophysiological purpose for the NRK2 expression and define how NRK2 contributes to NAD synthesis in the failing heart. In conclusion, our aims for this proposal will establish the role of NAD metabolism in heart failure and will help determine if targeting NAD-related pathways can be a viable treatment for patients.